摘要
让一种有抗菌活性的非致病性枯草芽孢杆菌BS224表达碱性成纤维细胞生长因子(bFGF),使其同时具有抗菌和促进伤口愈合的作用。用PCR方法克隆了一个α-淀粉酶高产株的α-淀粉酶基因的启动子和信号肽序列及3'端序列,合成了gnt(葡萄糖酸盐操纵元)终止子序列作为构建元件,和碱性成纤维细胞因子的cDNA序列一起插入质粒pHV32组建成整合型载体,该质粒来源于大肠杆菌质粒pBR322和枯草杆菌质粒pC194。然后通过原生质体转化法转化BS224,氯霉素抗性筛选得到转化体,用α-淀粉酶分析实验和Southern杂交证明其发生了同源重组,Western杂交证明碱性成纤维细胞因子有表达。
To make a kind of nonpathogenic Bacillus subtilis BS224with the antibacterial characteristics to express and secret basic fibroblast growth factor(bFGF)so that it has both antibacterial and accelerating wound healing na-ture.We constructed an integrative vector with theα-amylase promoter and signal sequence coding region obtained by PCR from the chromosome of anα-amylase hyperproducing strain,and the synthesized gnt (gluconate)terminator,and the3'sequence ofα-amylase gene inserting into plasmid pHV32.The bFGF cDNA was inserted into the vector.BS224was transformed by protoplast transformation.The transformants were screened by chloromycetin resistance and they were verified to integrate at the amy locus of BS224byα-amylase secretion analysis and Southern hybridiza-tion.Basic fibrobast growth factor expression was tested by Western immunoblot analysis.
出处
《生物技术通讯》
CAS
2002年第6期439-442,共4页
Letters in Biotechnology
